Screening for papilloma viruses

ABSTRACT

The invention relates to a method of screening for precursor lesions which can lead to cervical malignancy, methods of detecting and typing human papilloma virus infections, and reagents of use in these methods.

This application is a continuation of Ser. No. 09/314,268, filed May 18,1999, now U.S. Pat. No. 6,346,377 issued Feb. 12, 2002, which is acontinuation of international application No. PCT/GB97/03321, whichdesignated the United States and was filed Dec. 3, 1997, published inEnglish and which claims priority to foreign application GB9718745.4,filed Sep. 5, 1997, and GB9625142.6, filed Dec. 3, 1996.

FIELD OF THE INVENTION

This invention relates to a method of screening for precursor lesionswhich can lead to cervical malignancy, methods of detecting and typingHPV infections, and reagents of use in the above methods.

BACKGROUND OF THE INVENTION

Papillomaviruses (PVs) cause epithelial rumours in humans which vary inseverity depending on the site of infection and the HPV (human papillomavirus) type involved (Laimins-, 1993; Villiers de, 1994). Low risk typessuch as HPV 1 or HPV63 (Egawa et al. 1993a: Egawa et al. 1993b) causebenign cutaneous warts which progress to malignancy only rarely, whilehigh risk viruses such as HPV 16 and HPV31 cause flat warts at mnucosalsites, and are associated with high grade cervical intraepithelialneoplasia (CIN) and cancer (Schneider, 1994). Formation of anHPV-induced tumour is thought to require infection of an epithelialbasal cell, and the expression of viral early proteins in order tostimulate cell proliferation. The late stages of the virus life cycle,which ultimately lead to the production of infectious virions, areinitiated only as the infected cell migrates through the upperdifferentiated layers of the epidermis. Viral and cellular events whichinfluence HPV late gene expression have not been characterised as. untilrecently, there has been no convenient system for mimickina productiveinfection in vitro (Laimins, 1993)

Studies on naturally-occurring warts have revealed the virus to encodethree late proteins—L1 and L2, which are virion coat proteins (Doorbaret al, 1987), and E1^E4, a non-structural late protein of unknownfunction (Doorbar et al, 1986). In HPV1-induced warts the E1^E4 proteinis first expressed in cells of the lower spinous layer, and assemblesinto distinctive cytoplasmic and nuclear inclusions. During terminaldifferentiation it is post-transcriptionally modified by phosphorylation(Grand et al, 1989) and by removal of sequences from the N-terminus(Doorbar et al, 1988; Roberts et al, 1994). The E1^E4 proteins of highrisk viruses have been poorly characterised, because it has been thoughtthat HPV16-induced lesions contain only small numbers of productivelyinfected cells, and that these contain only low levels of E4 (Doorbar etal, 1996b: Crum et al, 1990). A single Mab (TVG 402) to HPV16 E1^E4 hasbeen used to locate the protein to the cytoplasm but was reported not towork well on paraffin-embedded archival material (Doorbar et al, 1992).Furthermore, polyclonal antibody studies on the E4 proteins of mucosalviruses have yielded conflicting results. One study has supported theabove findings (Crum et al, 1990), while another has indicated that theprotein is located to the nucleus (Palefsky et al. 1991).

In many countries there are screenig prograrmes to detect the presenceof cervical carcinoma at an early stage. Generally such prdgrammesoperate by obtaining cervical smears from women potentially at risk ofdeveloping cervical cancer, with the resulting smears routinelv beinzexamined bv conventional histopathological techniques. These techniquesare laborious and time-consuming, require considerable experience tointerpret results correctly, and frequently give rise to relativelylarge percentages of false positive results, causing unnecessary alarm.False negatives can occur when screening is carried out by inexperiencedpersonnel and can lead to the classification of pre-cancerous lesions asnormal. There is thus a need for an improved cervical cancer screeningmethod.

It is well known that there is a very strong correlation betweenHPV-infection and development of cervical carcinoma: over 90% of womenwith cervical carcinoma show evidence of HPV infections of the cervix.Accordingly, one possible alternative to conventional histopathologicalexamination of cervical smears is to examine samples for evidence of HPVinfection. For example, there have been numerous proposals to screen forcervical carcinoma by performing DNA hybridisation assays on samples,using nucleic acid probes specific for HPV sequences. Such hybridisationassays are generally favoured by those skilled in the art, because ofthe ready availability of suitable reagents and because of their highspecificity.

Thus, for example in Fields Virology (Fields et al, [Eds.] Virology Vol.2, p 2099, 3rd Edn. (1996) Raven Press, New York), an authoritativevirology text book, it is stated that “Diagnosis of an HPV type in atissue requires nucleic acid hybridization studies”.

In contrast, screening for cervical carcinoma by detection of expressionof HPV polypeptides has generally been disregarded, being consideredunsuitable for a number of reasons, primarily because of the difficultyin obtaining suitable reagents and, more significantly, many HPVsproduce very little virus protein in mucosal infections, makingdetection difficult, uncertain and unreliable. Thus, in Fields Virology(cited above) it is state that “immunologic detection of viral capsidantigens” is “of limited value”. The possibility of immunologicdetection of other viral antigens is not even considered. If one were todevelop a screening method based on detection of expression of viralproteins, the most likely choice of target would be those proteins whichare best-characterised, such as L1 or L2. The function of E4 protein isat present unknown. Its expression pattern in cervical lesions has notbeen determined conclusively in the prior art so the molecule has notbeen an obvious choice for selection as a target for detecting HPVinfection.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been demonstratedthat HPV infection can be detected in a sample taken from a patient byusing molecules which bind specifically to E4 protein of HPVs. Inparticular, the invention provides a method of screening samples forpre-cancerous cervical lesions, using molecules which bind specificallyto HPV E4 protein.

The present studies have clearly demonstrated HPV16 E4 protein to becytoplasrnic, and to be produced in cells supporting vegetative viralDNA replication.

In a first aspect the invention provides a method of detecting apapilloma virus infection in an organism, the method comprising thesteps of: obtaining a sample of the organism's cells from the site ofpotential papilloma virus infection; contacting the cells with amolecule that binds specifically to papilloma virus E4 protein; andmonitoring said binding.

In particular, the invention provides a method of screening forpre-cancerous cervical lesions, comprising the steps of: obtaining asample of cervical cells from a subject; contacting the cells with amolecule that binds specifically to HPV E4 protein; and monitoring saidbinding.

Moreover, the invention provides a method of determining the type(s) ofHPV infection in a patient, the method comprising the steps of:obtaining a sample of the patient's cells from the site of HPVinfection; contacting the cells with a molecule that binds specificallyto a subset of HPV E4 proteins; and monitoring said binding.

In a further aspect the invention provides an antibody molecule or anantigen-binding variant thereof, which binds specifically to HPV E4protein in the region of amino acid residues RPIPKPSPWAPKKHRRLSDQDSQTP(SEQ ID NO: 4) of HPV16 E4 protein, or the corresponding hydrophilicacid/base-rich region of other HPV E4 proteins.

The invention moreover concerns the use of molecules capable of bindingto E4 to target antiviral agents capable of destroying papilloma virusesand/or cells infected bv papilloma viruses. Such molecules may beantibodies or peptides as described above and exemplified herein,optionally conjugated to anticancer or antiviral agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid sequence of HPV16 E4 protein and thebinding sites of various antibody molecules or E4-specificantigen-binding fragments of antibodies; octapeptide sequences areidentified as follows: MADPAAAT, SEQ ID NO:5; ADPAAATK, SEQ ID NO:6;DPAAATKY, SEQ ID NO:7; PAAATKYP, SEQ ID NO:8; AAATKYPL, SEQ ID NO:9;AATKYPLL, SEQ ID NO:10; ATKYPLLK, SEQ ID NO:11; TKYPLLKL, SEQ ID NO:12;KYPLLKLL, SEQ ID NO:13; YPLLKLLG, SEQ ID NO:14; PLLKLLGS. SEQ ID NO:15;LLKLLGST, SEQ ID NO:16; LKLLGSTW, SEQ ID NO:17; KLLGSTWP, SEQ ID NO:18;LLGSTWPT, SEQ ID NO:19; LGSTWPTT, SEQ ID NO:20; GSTWPTTP, SEQ ID NO:21;STWPTTPP, SEQ ID NO:22; TWPTTPPR, SEQ ID NO:23; WPTTPPRP, SEQ ID NO:24;PTTPPRPI SEQ ID NO:25; TTPPRPIP, SEQ ID NO:26; TPPRPIPK, SEQ ID NO:27;PPRPIPKP, SEQ ID NO:28; PRPIPKPS, SEQ ID NO:29; RPIPKPSP, SEQ ID NO:30;PIPKPSPW, SEQ ID NO:31; IPKPSPWA, SEQ ID NO:32; PKPSPWAP, SEQ ID NO:33;KPSPWAPK, SEQ ID NO:34; PSPWAPKK, SEQ ID NO:35; SPWAPKKH, SEQ ID NO:36;PWAPKKHR, SEQ ID NO:37; WAPKKHRR, SEQ ID NO:38; APKKHRRL, SEQ ID NO:39;PKKHRRLS, SEQ ID NO:40; KKHRRLSS, SEQ ID NO:41; KHRRLSSD, SEQ ID NO:42;HRRLSSDQ, SEQ ID NO:43; RRLSSDQD, SEQ ID NO:44; RLSSDQDQ, SEQ ID NO:45;LSSDQDQS, SEQ ID NO:46; SSDQDQSQ, SEQ ID NO:47; SDQDQSQT, SEQ ID NO:48;DQDQSQTP, SEQ ID NO: 49; QDQSQTPE, SEQ ID NO:50; DQSQTPET, SEQ ID NO:51;QSQTPETP, SEQ ID NO:52; SQTPETPA, SEQ ID NO:53; QTPETPAT, SEQ ID NO:54;TPETPATP, SEQ ID NO:55; PETPATPL, SEQ ID NO:56; ETPATPLS, SEQ ID NO:57;TPATPLSC, SEQ ID NO:58; PATPLSCC, SEQ ID NO:59; ATPLSCCT, SEQ ID NO:60;TPLSCCTE, SEQ ID NO:61; PLSCCTET, SEQ ID NO:62; LSCCTETQ, SEQ ID NO:63;SCCTETQW, SEQ ID NO:64; CCTETQWT, SEQ ID NO:65; CTETQWTV; SEQ ID NO:66;TETQWTVL, SEQ ID NO 67; ETQWTVLQ, SEQ ID NO:68; TQWTVLQS, SEQ ID NO:69;QWTVLQSS, SEQ ID NO:70; WTVLQSSL, SEQ ID NO:71; TVLQSSLH, SEQ ID NO:72;VLQSSLHL, SEQ ID NO:73; LQSSLHLT, SEQ ID NO:74; QSSLHLTA, SEQ ID NO:75;SSLHLTAH, SEQ ID NO:76; SLHLTAHT; SEQ ID NO: 77; LHLTAHTK, SEQ ID NO:78;HLTA HTKD, SEQ ID NO: 79; LTAHTKDG, SEQ ID NO:80; TAHTKDGL, SEQ IDNO:81; AHTKDGLT, SEQ ID NO:82, HTKDGLTV, SEQ ID NO:83; TKDGLTVI, SEQ IDNO:84; KDGLTVIV, SEQ ID NO:85; DGLTVIVT, SEQ ID NO:86; GLTVIVTL, SEQ IDNO:87; LTVIVTLH, SEQ ID NO:88; and TVIVTLHP, SEQ ID NO:89.

FIG. 1B shows the sequence of the E4 protein from HPV16 (top row, SEQ IDNO:90), HPV1 (bottom row, SEQ ID NO:91) and a consensus sequence (middlerow, SEQ ID NO:92), and the binding sites of various antibodies orantigen-binding variants of antibodies;

FIGS. 2A–2D show four sensograms (arbitrary response units against timein seconds) obtained using surface plasmon resonance apparatus;

FIGS. 3(A–D) show the use of synthetic Fabs to localize HPV16 E4 proteinin vivo by immunostaining of a low-grade HPV16 CIN I. p FIGS. 4(A–I)show the results of staining for L1, HPV16 E4, HPV63 E4, and DAPI, inlow-grade HPV16-induced lesion (CIN I), high grade HPV16-induced lesion(CIN II/III), and a section through a verruca caused by HPV63.

FIGS. 5(A–F) show the results of staining for HPV16 E4 and biotinylatedDNA probe, in low grade HPV16 lesions and in benign cutaneous warts.

FIGS. 6(i) (A–F) show the results of staining, for HPV16 E4, HPV1 E4,HPV63 E4, differentiation specific mucosal keratins or cutaneouskeratins, and DAPI counter stain, in a HPV16-induced CIN I, anHPV-1-induced verruca, and an HPV63-induced wart.

FIGS. 6(ii) (A–I) show the results of staining for HPV16 E4, HPV1 E4,HPV63 E4, filaggrin, and DAPI counter stain, in a HPV16-induced CIN I,an HPV-1-induced verruca, and an HPV63-induced wart

FIGS. 7(A–D) show the association of HPV16 E4 proteins with perinuclearbundles and filamentous structures in vivo.

FIGS. 8(A–C) show staining in the upper layers of a HPV16 CIN for anepitope in the C-terminal half of the E4 protein, the N-terminal 12amino acids of the HPV16 E1 E4 protein, or DAPI.

FIG. 9 is an amino acid sequence alignment of part of HPV E4 proteins.

Sequences are identified as follows: SEQ ID NO: 93; HPV54, SEQ ID NO:94; HPV32, SEQ ID NO: 95; HPV42, SEQ ID NO: 96; HPV3, SEQ ID NO: 97;HPV28, SEQ ID NO: 98; HPV10, SEQ ID NO:99; HPV29, SEQ ID NO: 100; HPV61,SEQ ID NO:101; HPV2a, SEQ ID NO:102; HPV 27, SEQ ID NO:103; HPV57, SEQID NO:104; HPV26, SEQ ID NO:105; HPV30, SEQ ID NO:106; HPV53, SEQ IDNO:107; HPV56, SEQ ID NO:108; HPV66, SEQ ID NO:109; HPV18, SEQ IDNO:110; HPV45, SEQ ID NO:111, HPV39, SEQ ID NO: 112; HPV70, SEQ ID NO:113; HPV59, SEQ ID NO:114; HPV7, SEQ ID NO: 115; HPV40, SEQ ID NO: 116;HPV16, SEQ ID NO: 117; HPV35, SEQ ID NO: 118; HPV31,SEQ ID NO: 119;HPV52, SEQ ID NO: 120; HPV33, SEQ ID NO: 121; HPV58, SEQ ID NO: 122;RHPV1, SEQ ID NO: 123; HPV66, SEQ ID NO: 124; HPV11, SEQ ID NO: 125;HPV44, SEQ ID NO: 126; HPV55, SEQ ID NO: 127; HPV13, SEQ ID NO: 128;PCPV1, SEQ ID NO: 129; HPV34, SEQ ID NO: 130; HPV19, SEQ ID NO: 131;HPV25, SEQ ID NO: 132; HPV20, SEQ ID NO: 133, HPV21, SEQ ID NO: 134;HPV14d, SEQ ID NO: 135; HPV5, SEQ ID NO: 136; HPV36, SEQ ID NO: 137;HPV47, SEQ ID NO: 138; HPV12, SEQ ID NO: 139; HPV8, SEQ ID NO: 140;HPV24, SEQ ID NO: 141; HPV15, SEQ ID NO: 142; HPV17, SEQ ID NO: 143;HPV37, SEQ ID NO: 144; HPV9, SEQ ID NO: 145; HPV22, SEQ ID NO: 146;HPV23, SEQ ID NO: 147; HPV38, SEQ ID NO: 148; HPV49, SEQ ID NO: 149;HPV4, SEQ ID NO: 150; HPV65, SEQ ID NO: 151; HPV48, SEQ ID NO: 152;HPV50, SEQ ID NO:153; HPV60, SEQ ID NO:154; BPV1, SEQ ID NO: 155; BPV2,SEQ ID NO:156; EEPV, SEQ ID NO:157; DPV, SEQ ID NO:158; BPV4, SEQ IDNO:159; HPV41, SEQ ID NO:160; COPV, SEQ ID NO:161; CRPV,SEQ ID NO: 162;ROPV, SEQ ID NO: 163; HPV1a, SEQ ID NO: 164; HPV63, SEQ ID NO:165; andMnPV, SEQ ID NO: 166.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the present invention permits the detection,identification and diagnosis of papilloma viruses and papilloma virusinfections in organisms susceptible to such infections.

Such organisms are preferably mammals, and most preferably humans. Wherethe organism is a human organism, the papilloma virus may be a type ortypes of human papilloma virus (HPV).

The sample of patient's cells may comprise skin cells (e.g. in the caseof warts, veruccas and the like, caused by cutaneous HPV infections).Cutaneous lesions, such as those induced by HPV types 5, 8, 14, 17, 20,are difficult to manage clinically, and are often associated withmalignancies in imnmunlosuppressed patients (Benton et al, 1992Papillomavinis Reports 3, 23–26). Alternatively, the sample may comprisemucosal cells, especially cervical cells, In the case of HPV infectionsof the urinogenital tract. Methods of obtaining and preparinge suchsamioples for use in the method of the invention are known to thoseskilled in the art or will be apparent from the present disclosure.

The term “pre-cancerous cervical lesions” is intended to refer to thoseabnormalities which clinically may be described as “pre-malignant”conditions and which may, without treatment, proceed to fullmalignancies. As set forth above, such lesions are screened forroutinely by, for example, cervical smear testing. The present inventionallows for cells obtained from patients by methods such as cervicalsmears to be tested more accurately and more quickly for HPV infection.

Preferably, the molecule which binds specifically to E4 proteincomprises an antibody motecule or an antigen-binding variant thereof,such as an Fab, Fv, scFv, “diabody” and the like. The molecule maycomprise monoclonal or polyclonal antibodies, or antigen-bindingportions of antibodies selected from libraries by screening (e.g. usingphage display technology). Alternatively the molecule may be some otherpolypeptide, peptide, a synthetic compound or an RNA or DNA aptamer,generated by a procedure such as SELEX. In some preferred embodimentsthe molecule comprises a label moiety, such as a fluorophore,chromophore, enzyme or radio-label, so as to facilitate monitoring ofbinding of the molecule to E4 protein. Such labels are well-known tothose skilled in the art and include, for example, fluoresceinisothiocvanate (FITC), β-galactosidase, horseradish peroxidase,streptavidin, biotin, ³⁵S or ¹²⁵I. Other examples will be apparent tothose skilled in the art. The label may in some instances be conjugatedto the antibody or antigen-binding variant, or may be present (where thelabel is a peptide or polypeptide) as a fusion protein.

Prefefrably the molecules used in the method of the invention bindselectively to the E4 protein of a certain HPV type or types, but nottothe E4 protein of other HPV types. Accordingly, in one embodiment theinvention can be used to determine the type or types of HPV infecting apatient. This is very significant, as progression to malignant disease(and hence clinical prognosis) is heavily dependent on HPV type.Accordingly, in a second aspect the invention provides a method ofdetermining the type(s) of HPV infection in a patient, the methodcomprising the steps of: obtaining a sample of the patient's cells fromthe site of HPV infection: contacting the cells with a molecule thatbinds specifically to a subject of HPV E4 proteins; and monitoring saidbinding.

In the method of the second aspect of the invention, the subset of E4proteins to which the molecule binds may consist of a single HPV type E4protein, or may consist of a plurality of E4 proteins of differenttypes, but will not encompass the E4 proteins of all known HPV types,such that binding or non-bindiny (as appropriate) of the molecule to theE4 protein present in the cell sample will allow an investigator to makecertain deductions about the identity of the HPV type(s) infecting thepatient.

In practice it may be advantageous to employ a plurality of differentmolecules, which bind to different subsets of E4 proteins. This may benecessary to identify unambiguously the type(s) of HPV infecting thepatient, although it may not be essential as a prognostic indicator. Forexample, the ability to limit the infecting HPV type(s) to a particularsubset (or exclude such a subset) may be sufficient. By way ofexplanation, it is known that mucosal HPV types 6, 11, 42, 43 and 44 areassociated with external genital papillomas (condylomata accuminata)which have a low risk of progression to cancer, but are difficult toeradicate and are disruptive to the lives of the patients. The hiherrisk mucosal types (31, 33, 35, 51, 52, 58, 61 and 16, 18, 45, 56) causeasymptomatic flat warts (flat concyloma) which can progress to highgrade cervical intraepithelial neoplasia (CIN) and cancer. The highestrisk of progression to malignancy is associated with lesions caused byHPV types 16, 18, 45 and 56.

Molecules which bind to desired HPV types, but not to undersired HPVtypes, may be generated for example by randomisation and selectiontechniques. These include phage display, and other techniques suitablefor displaying antibodies or other polypeptides; and procedures forgenerating nucleic acid binding molecules, for example RNA aptamers,such as SELEX. These procedures are well known to those of ordinaryskill in the art and described below for the purposes ofexemplification. The invention accordingly provides HPV-bindingmolecules targetted to the HPV E4 protein, which are useful in methodsas described herein.

According to the present invention, E4-binding molecules are preferablytargeted to extracellular portions of the E4 polypeptide. Such portionstend to be hydrophilic in character. Preferably, therefore, the E4binding molecules according to the invention specifically bind tohydrophilic portions of the HPV E4 protein.

The present invention moreover provides a particular region of the E4protein to which 10 molecules (particularly antibody molecules orvariants thereof) may bind with considerable specificity. Althoughhomologous regions exist in all HPV E4 proteins, the region varies inamino acid sequence between HPVs of different types. The regioncorresponds to a peak of hydrophilicity in the E4 protein and isprobably surface-exposed. The region is highly charged (acid/base-rich).In HPV type 16, the amino acid sequence of the region is (fromN-terminal to C-terminal) RPIPKPSPWAPKKHRRLSDQDSQTP (SEQ ID NO: 4).Clearly the amino acid sequence of the E4 proteins of other HPV typeswill not necessarily be identical to that in type 16, but with thebenefit of the present disclosure (e.g. FIG. 9) the corresponding regioncan readily be identified in other E4 proteins by those skilled in theart by use of conventional alignment and sequence comparison computerprograms (about 65 of the 70 or so known HPV genomes have been clonedand sequenced).

Thus, in a third aspect the invention provides an antibody molecule, oran antigen binding variant thereof, which binds specifically to HPV E4protein in the region of amino acid residues RPIPKPSPWAPKKHRRLSDQDSQTP(SEQ ID NO: 4) of HPV16 E4 protein, or the corresponding hydrophilic,acid/base-rich region of other HPV E4 proteins, preferably other thanthe antibody TVG 402 identified by Doorbar et al, (1992 Virology 187,353–359).

Moreover, the invention provides the use of an antibody molecule, or anantigen binding variant thereof, which binds specifically to HPV E4protein in the region of amino acid residues RPIPKPSPWAPKKHRRLSDQDSQTP(SEQ ID NO: 4) of HPV16 E4 protein, or the corresponding hydrophilic,acid/base-rich region of other HPV E4 proteins for the detection of HPVinfections as described herein.

The corresponding hydrophilic acid/base-rich regions of large numbers ofdifferent HPV types are shown in FIG. 9. FIG. 9 shows a consensus-typeamino acid sequence (“most likely”) on the top row, with the sequence ofHPV E4 proteins below. Dots indicate gaps introduced to facilitate thealignment, dashes denote amino acid residue matches with the consensussequence. Numbering on the right hand side of the figure indicates thenumber of amino acid residues from the actual or predicted E1^E4 splicesite. It will be appreciated by those skilled in the art from thealigrment that whilst the hydrophilicity of the reaion is conservedamongst different HPV types, the actual amino acid sequence varies quiteconsiderably, such that reagents binding to this region may be expectedto be highly HPV type-specific.

Preferably the antibody of the invention has a binding site, asidentified by the SPOTS epitope mapping system within the region.RPIPKPSPWAPKKHR (SEQ ID NO: 167) (or the corresponding amino acidsequence from other HPV types). A particularly preferred molecule is theFab fragment TVG405, described further below, which binds to the epitopePKPSPWAPKKH(R) (SEQ ID NO: 168) with extremely high affinity and is ofparticular usefulness in the methods of the invention defined above.

The arginine residue indicated in brackets at the C-terminal of theTVG405 epitope is not essential for high affinity binding.

The Fab fragment TVG405 was isolated by the present inventor using phagedisplay technology, as described below. Those skilled in the art willunderstand that different antibodies or Fab fragments may readily beobtained by using similar phage display techniques (and screening withE4 proteins or portions thereof), or by using more conventionalimmunisation techniques (e.g. immunising mice, rabbits, rats or the likewith E4 protein or peptides corresponding to portions of the E4 protein)to obtain polyclonal antisera or monoclonal antibodies (using well knownhybridoma techniques of Milstein et al). Complete antibody molecules canreadily be prepared from Fab—encoding sequences (e.g. isolated by phagedisplay techniques) using standard DNA manipulation techniques describedby Sambrook et al, (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press, NY, USA) to join appropriate DNAsequences.

Similarly, standard DNA manipulative techniques can be used to modifyDNA sequences encoding anti-E4 antibodies or antigen-binding variantsthereof. In particular site-directed mutagenesis or PCR can be used tomodify the coding sequences, so as to produce modified anti-E4antibodies with different binding specificities or affinities.Alternatively fusion proteins, comprising the E4-binding site of an Fab,Fv or antibody and the like, may be prepared.

Molecules capable of binding E4 may be used as anti-viral or anti-canceragents, or parts of such agents. For example, antibody molecules orE4-binding peptide as described above may be employed for this purpose.Preferably, however, the E4 protein and/or molecules capable of bindingthereto may be used to design E4-binding molecules, preferably smallmolecules, by rational drug design.

Such a process preferably involves the crystallisation of E4 or amolecule capable of binding thereto. More preferably, such a processinvolves the co-crystallisation of E4 and a binding agent. Such aprocedure gives information concerning the interaction between E4 andthe binding molecule, which can be used to design small moleculescapable of mimicking the binding interaction.

Crystallisation involves the preparation of a crystallisation buffer,for example by mixing a solution of the peptide or peptide complex witha “reservoir buffer”, preferably in a 1:1 ratio, with a lowerconcentration of the precipitating agent necessary for crystalformation. For crystal formation, the concentration of the precipitatingagent is increased, for example by addition of precipitating agent, forexample by titration, or by allowing the concentration of precipitatingagent to balance by diffusion between the crystallisation buffer and areservoir buffer. Under suitable conditions such diffusion ofprecipitating agent occurs along the gradient of precipitating agent,for example from the reservoir buffer having a higher concentration ofprecipitating agent into the crystallisation buffer having a lowerconcentration of precipitating agent. Diffusion may be achieved forexample by vapour diffusion techniques allowing diffusion in the commongas phase. Known techniques are, for example, vapour diffusion methods,such as the “hanging drop” or the “sitting drop” method. In the vapourdiffusion method a drop of crystallisation buffer containing the proteinis hanging above or sitting beside a much larger pool of reservoirbuffer. Alternatively, the balancing of the precipitating agent can beachieved through a semipermeable membrane that separates thecrystallisation buffer from the reservoir buffer and prevents dilutionof the protein into the reservoir buffer.

In the crystallisation buffer the peptide or peptide/binding partnercomplex preferably has a concentration of up to 30 mg/ml, preferablyfrom about 2 mg/ml to about 4 mg/ml.

Formation of crystals can be achieved under various conditions which areessentially determined by the following parameters: pH, presence ofsalts and additives, precipitating agent, protein concentration andtemperature. The pH may range from about 4.0 to 9.0. The concentrationand type of buffer is rather unimportant, and therefore variable, e.g.in dependence with the desired pH. Suitable buffer systems includephosphate, acetate, citrate, Tris, MES and HEPES buffers. Useful saltsand additives include e.g. chlorides, sulphates and further saltsspecified in Example 1. The buffer contains a precipitating agentselected from the group consisting of a water miscible organic solvent,preferably polyethylene glycol having a molecular weight of between 100and 20000, preferentially between 4000 and 10000, or a suitable salt,such as a sulphates, particularly ammonium sulphate, a chloride, acitrate or a tartrate.

A crystal of E4 itself or an E4-derived peptide, or E4 (peptide)/bindingpartner complex according to the invention may be chemically modified,e.g. by heavy atom derivatization. Briefly, such derivatization isachievable by soaking a crystal in a solution containing heavy metalatom salts, or a organometallic compounds, e.g. lead chloride, goldthiomalate, thimerosal or uranyl acetate, which is capable of diffusingthrough the crystal and binding to the surface of the protein. Thelocation(s) of the bound heavy metal atom(s) can be determined by X-raydiffraction analysis of the soaked crystal, which information may beused e.g. to construct a three-dimensional model of the peptide.

A three-dimensional model is obtainable, for example, from a heavy atomderivative of a crystal and/or from all or part of the structural dataprovided by the crystallisation. Preferably building of such modelinvolves homology modelling and/or molecular replacement.

The preliminary homology model can be created by a combination ofsequence alignment with any of the E4 proteins the sequence of which isknown, secondary structure prediction and screening of structurallibraries. For example, the sequences of HSV 16 and 34 E4 can be alignedas set forth herein.

Computational software may also be used to predict the secondarystructure of E4 peptides or peptide complexes. The peptide sequence maybe incorporated into the E4 structure. Structural incoherences, e.g.structural fragments around insertions/deletions can be modelled byscreening a structural library for peptides of the desired length andwith a suitable conformation. For prediction of the side chainconformation, a side chain rotamer library may be employed.

The final homology model is used to solve the crystal structure of E4 orpeptides thereof by molecular replacement using suitable computersoftware. The homology model is positioned according to the results ofmolecular replacement, and subjected to further refinement comprisingmolecular dynamics calculations and modelling of the inhibitor used forcrystallisation into the electron density.

Similar approaches may be used to crystallise and determine thestructure of E4-binding polypeptides, including antibodies and antibodyfragments, for example those provided by the present invention.

It has surprisingly been found that E4 expression correlates stronglywith vegetative DNA replication in HPV-infected cells, making detectionof E4 expression a particularly appropriate indicator of HPV infection,and thus particularly useful in screening for precancerous cervicallesions.

Present available methods of cervical screening by HPV detection arebased on DNA hybridisation. They involve cell lysis or permeabilisationand are performed in an ELISA-type 96 well format. The hybridisation isultimately visualised as a colour change in one of the wells.

Although the antibodies of the present invention could be used in asimilar way (i.e. following cell lysis), they are amenable to a quickerprocedure which would be more readily carried out routinely byhistopathology laboratories. Samples comprising cervical cells may betaken as usual. These are be spread for example on a microscope slide orother support using techniques known in the art, for example asexemplified herein, and stained with, for example, an anti-E4 Fab.Detection may be performed with a secondary antibody-enzyme conjugate(horseradish peroxidase, alkaline phosphatase), or the Fab could bedirectly conjugated, for example to a fluorophore, such as FITC. Thisapproach may be adapted for use with systems that are currentlyavailable for increasing the sensitivity of antibody detection. Atpresent, cervical smears are examined routinely by microscopy. Theproposed approach would require no new equipment and could easily fitaround existing methods.

It is envisaged that the standard method of detection may be modified.Antibody binding may be carried out while the cells are in suspension,with cells being spun down prior to analysis. This would improve thequality of the screen.

Considerable effort in diagnosis is aimed at automating screeningmethods. The use of antibodies or antigen-binding variants thereof forHPV detection greatly facilitates this.

In summary, it has been shown that:

-   -   1. The E4 protein can be detected in productively infected        HPV-induced lesions, and in low and high grade cervical        neoplasia even when differentiation of the infected keratinocyte        is insufficient to support production of capsid proteins and        assembly of infections virions.    -   2. E4 expression correlates closely with vegetative viral DNA        replication indicating that detection of the E4 protein is as        efficient as detection of viral DNA replication for the        detection of virus infection.    -   3. The E4 protein is abundant in the upper layers of infected        tissue and is thus detectable in cells taken during routine        smear tests.

The invention will now be described by way of illustrative examples.

EXAMPLE 1 Preparation of Anti-E4 Monoclonal and PolyclonalImmunoglobulins

Although Mabs against HPV16 E1^E4 have been described previously(TVG401, 402, 403; Doorbar et al, 1992) these reagents recognise asingle overlapping epitope at the major antigenic site of E4, and havebeen reported not to detect the protein in archival tissue biopsies(Doorbar et al, 1992).

Although these results suggest that E4 may not be a candidate forimmunological detection of HPV, further antibodies are generatedtargeted at the N and C termini of HPV16 E4.

The generation of further Mabs by standard hybridoma technology resultsin the isolation of TVG404, an IgM which recognises an epitope at thevery C-terminus of the protein.

To complement this reagent polyclonal antiserum to the N-terminus of theprotein is raised against an N-terminal synthetic peptide (−E4 N term).Polyclonal antibodies (to HPV16 and HPV63 E4 proteins) are prepared byimmunisation of rabbits with maltose binding protein E4 fusion protein(MBP-E4). Antibody titres are monitored in ELISA using purifiedglutathione S transferase E4 fusion protein (GST-E4).

Antibodies to the N-terminus of the protein are raised against thesynthetic peptide MADPAAATKYPLC (SEQ ID NO: 169) after conjugation tothyroglobulin or keyhole limpet haemocyanin through its C-terminalcysteine residue. Conjugation is carried out usingm-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as described byGreen et al (1982).

Antibody specificities are confirmed by epitope mapping, as follows: theHPV 16 E4 protein is synthesised as a series of 85 overlapping octamers(single amino acid overlap) by solid phase fmoc chemistry using theSPOTS epitope mapping system (Genosys Biotechnologies, Cambridge, UK).Accuracy of synthesis is confirmed using the HPV16 E1^E4 monoclonalTVG402 which binds the major antigenic site of the protein (Doorbar etal, 1992). Filters are regenerated as described by the manufacturers andantibody binding is visualised by ECL (Amersham, Little Chalfont, UK).Polyclonal serum is used at 1/250 dilution, purified Fabs atapproximately 1 g/ml, and hybridoma supernatant at 1/10 dilution.

In FIG. 1A the sequences of the 85 overlapping E4 synthetic peptides areshown at the top of the figure, and the results of the epitope mappingexperiments are shown below. The dark spots represent binding of theantibody to the synthetic peptide shown above it. Only the portion ofthe filter containing peptides which react with each antibody are shown.

In FIG. 1B, the locations of epitopes on the E1^E4 amino acid sequenceare summarised above the HPV16 sequence. Alignment with a consensus E4sequence prepared by comparison of 70 putative E1^E4 sequences (Doorbarand Myers, 1996b) is shown beneath the sequence of HPV16 E1^E4, and thesequence of the HPV1 E1^E4 protein is shown beneath this. The bindingsites of the existing HPV1 E1^E4 Mabs (Doorbar et al, 1988) are shownbeneath the HPV1 sequence. The proteolytic cleavage sites that give riseto the 16K and 10/11K gene products in the E1^E4 protein of HPV1(Doorbar et al, 1988; Roberts et al, 1994) are indicated beneath theHPV1 sequence allowing prediction of putative sites in the E1^E4sequence of HPV16.

EXAMPLE 2 Preparation of Synthetic Immunoglobulins

Fabs are isolated from a synthetic antibody displayed on fdbacteriophage (Griffiths et al, 1994) as described below. Immunotubes(Life Technologies, Paisley, UK) are coated overnight at 4° C. witheither MBP-E4 or GST-E4 at a concentration of 0.1 g/ml. These aresubsequently blocked at 37° C. for 1 hour in PBS/2% marvel™ prior toincubation in the presence of 10¹¹ phage on a blood tube rotator (37°C.). Unbound phage are poured off and tubes are washed 10× with PBS/0.1%Tween 20. Binders are eluted with 100 mM triethylamine pH 11.0 (1 ml)and immediately neutralised with 1M Tris (pH8.0) before beingreintroduced into E. coli TG1 cells. The enriched library is grown upand the selection procedure repeated three more times.

Phage selections are carried out alternately against GST 16 E1^E4 andMBP 16 E1^E4 in order to prevent isolation of antibodies to MBP or GSTprotein, using a repertoire of 6.5×10¹⁰ (Griffiths et al, 1994). MBP 16E4 is expressed at higher levels (>50 mg/litre of bacteria) than the GSTfusion (approx. 5 mg/litre of bacteria) but, in any event, antibodyisolation requires as little as 1 g of antigen (Hawkins et al, 1992).Phage displaying antibodies with affinity for E4 are identified by ELISA(against GST-E4, MBP-E4, GST and MBP), and activity is confirmed byphage western blotting. Immunoglobulin genes are transferred from theisolated phage into the bacterial expression vector pUC119.His.myc(Griffiths et al 1994) and soluble Fabs are purified from theperiplasmic space of induced bacteria by Nickel-NTA chromatography(Qiagen, Crawley UK). Antibody titres are established by ELISA.

After four rounds of selection, individual clones are examined for theirability to bind either E1^E4, unfused GST or MBP, or bovine serumalbumin (BSA). 47 clones (out of 100) are able to bind MBP 16 E1^E4, ofwhich 39 could also bind GST 16 E4. None of these clones interacted withBSA, GST or MBP. BstNI fingerprinting (Marks et al, 1992: Nissim et al,1994) revealed three distinct Fabs amongst these clones, and theirimmunoglobulin genes are subcloned into the prokaryotic expressionvector pUC119His.6myc to allow the production of soluble anti-E4 Fabs(Griffiths et al, 1994). Approximately 5 mg (per liter of bacteria) ofanti-E4 Fab (TVG 405, 406 and 407) can be extracted from the periplasmicspace of induced bacteria and all are found to specifically detect E1^E4by ELISA and western blotting. Fab TVG 407 binds an epitope which isidentical to that recognised by the hybridoma-derived Mab, TVG 409 (FIG.1). The remaining synthetic Fabs recognise novel epitopes upstream (TVG405) or downstream (TVG 407) of this major antigenic region of E4 andthe results are summarised in FIG. 1.

It is found that the amino acid sequence of the CDR3 loops of the TVG405 and TVG 407 Fabs are as follows:

TVG 405 heavy chain CDR3 sequence: LLRGAFDY (SEQ ID NO: 170) light chainCDR3 sequence: NSRDSSGGNAV (SEQ ID NO: 171) TVG 407 heavy chain CDR3sequence: LVQGSFDY (SEQ ID NO: 172) light chain CDR3 sequence: QADSSTHV(SEQ ID NO: 173)Measurement of Antibody Affinity

Affinities of synthetic (TVG405, TVG406 and TVG407), andhybridoma-derived Fabs (TVG402) are analysed by surface plasmonresonance using a BIAcore 2000 instrument (Pharmacia Biosensor, St.Albans, UK) as described by the manufacturer. MBP-E4 aggregates aredissociated under reducing conditions in 0.5% SDS, 1 mMβ-mercaptoethanol, 50 mM Na₂CO₃/NaHCO₃ (pH 8.5) and biotinylated usingNHS-LC-biotin (Sigma, St Louis, USA; 25 mg/ml in DMSO) at abiotin:protein molar ratio of 6:1 (Johnson et al, 1991). MonomericMBP-E4 is recovered by FPLC chromatography using a Superdex S200 HR10/30column run in 6M Urea/1 mM β-mercaptoethanol/PBS/0.2 mM EDTA (pH7 2),before being bound to a streptavidin-coated sensor chip and “refolded”in vitro in PBS/0.2 mM EDTA/0.1 mg/ml protease-free BSA (Sigma). Fabsare isolated from purified TVG402 using an Immunopure Fab kit (Pierce,Rockford, USA), and monomeric preparations are obtained by FPLC gelchromatography (Superdex S200 HR10/30 column run in PBS/0.2 mM EDTA (pH7.2)) Sensor chip surfaces are regenerated using 6M urea column buffer(described above). On and off rates are derived by non linear curvefitting using the proprietary ‘BIAanalysis’ software.

Binding activities are in the order of 20% of total protein levels forthe bacterially-derived antibodies, and 50% for Fabs derived fromhybridoma culture supernatant. The affinities of TVG405 and TVG402 arecalculated from on- and off-rates obtained by non-linear curve fittingto sets of BIAcore binding curves.

FIG. 2A shows an overlay of binding curves (sensograms) obtained afterpassing Fab TVG405 over a BIAcore chip coated with MBP-E4 fusion proteinas described above. Fab concentrations range from 10 mM (lowest curve)to 300 nM (upper curve) through 5 intermediate dilutions. The extent ofbinding is indicated in resonance units on the X-axis, against time inseconds on the Y-axis. Purified Fab is injected at around 100 secondsand washing initiated at 700 seconds. The affinity (K_(d)) of TVG405 iscalculated as between 0.3 and 1.25 nM by analysis of the association anddissociation curves using BIAevaluation software (Pharmacia, UK).

FIG. 2B shows an overlay of binding curves (as described above) for thehybridoma-derived Fab TVG402 over a concentration range 100 nM to 1 M.The K_(d) is estimated as 70 nM.

TVG405 has an association rate constant (k_(on)) of 1.8×10⁶ M⁻¹.s⁻¹ andan off rate (k_(off)) of 2×10³ s⁻¹ indicating a molar dissociationconstant (Kd) of approximately 1 nM. The best hybridoma-derivedantibody—TVG402—has an affinity of only 70 nM, and had a k_(on) of4.2×10⁴ M⁻¹.s⁻¹ and a k_(off) value of 3×10³ s⁻¹. TVG 406 and 407displayed rapid kinetics and are thus examined by Scatchard analysis ofequilibrium binding data, as shown for TVG407.

FIG. 2C shows the equilibrium binding curve of Fab TVG407, whichdisplays rapid kinetics. FIG. 2D shows Scatchard analysis of the datapresented in FIG. 2C using BIAevaluation software. Equilibrium valuesare corrected for bulk refractive index changes by subtracting valuesfrom a surface blocked with biotin, from the values shown in FIG. 2C. Inthe plot shown the slope is—K_(d) and the Y-axis intercept is ‘R_(max)’,i.e. the binding level at saturation with Fab. The uncorrected K_(d)values for TVG407 and TVG406 are 250 nM and 140 nM which, when theactivity of the Fab preparation is considered, indicates actualaffinities of 50 nM and 28 nM.

TVG407 has an affinity (Kd) of 50 nm after correction for biologicalactivity, and TVG406 has an affinity (Kd) of 28 nM. The amino acidsequence of the heavy and light chain CDR3 loops are established by DNAsequencing, further confirming that the three antibodies are distinct.

EXAMPLE 3 Preparation of Anti-E4 Peptides

A commercially available two-hybrid screening kit is purchased fromClonTech and employed for identifying naturally occurring E4-bindingpeptides, according to the instructions given by the manufacturer. AHeLa cDNA library, obtained from the same supplier, is screened. By thismethod, seven DNA sequences are isolated which encode E4-bindingpolypeptides, of which three are identified after sequencing.

The first peptide is ferritin. (SEQ ID NO: 1)

The second peptide is a keratin filament binding protein, which has thesequence set forth in (SEQ ID NO: 2).

The third polypeptide is a novel polypeptide recognised as a member ofthe DEAD box family of proteins, which contain the characteristicsequence motif DEAD (SEQ ID NO: 129). The sequence of the thirdpolypeptide is shown in (SEQ ID NO: 3).

In order to identify the site of interaction between these polypeptidesand E4, a series of overlapping peptides of between 10 and 20 aminoacids in length is generated by PCR and displayed on phage as describedabove. The binders are subsequently employed as screening agents toidentify HPV16 in mucosal lesions.

EXAMPLE 4 Preparation of Anti-E4 RNA Oligonucleotides

RNA oligonucleotides, known as aptamers, which are capable of specificbinding to target molecules can be generated by selection proceduressuch as SELEX. The SELEX method involves selection of nucleic acidaptamers, single-stranded nucleic acids capable of binding to a desiredtarget, from a library of oligonucleotides. Starting from a library ofnucleic acids, preferably comprising a segment of randomised sequence,the SELEX method includes steps of contacting the library with thetarget under conditions favourable for binding, partitioning unboundnucleic acids from those nucleic acids which have bound specifically totarget molecules, dissociating the nucleic acid-target complexes,amplifying the nucleic acids dissociated from the nucleic acid-targetcomplexes to yield a ligand-enriched library of nucleic acids, thenreiterating the steps of binding, partitioning, dissociating andamplifying through as many cycles as desired to yield highly specific,high affinity nucleic acid ligands to the target molecule.

DNA Oligonucleotide Library

DNA oligonucleotides 73 bases in length, having a randomised portion of26 bases, are used for the development of an aptamer capable of bindingE4. A library of synthetic RNA oligonucleotides having the followingstructure is prepared:

5′ CCTGTTGTGAGCCTCCTGTCGAA(26N)TTGAGCGTTTATTCTTGTC TCCC 3′ (SEQ ID NO:174)

Where N stands for any possible base in the random region. The randomregion is generated by using a mixture of all four nucleotides (ratio6:5:5:4, A:C:G:T, to allow for differences in coupling efficiency)during the synthesis of each nucleotide in that stretch of theoligonucleotide library. The resulting complexity is theoretically 4²⁶molecules. The scale of synthesis (0.1 μmol) followed by gelpurification yields 8.8 nmol which puts an absolute upper limit ofapproximately 5×10¹⁵ on the number of different molecules actuallypresent.

PCR Amplification with a 5′ primer that introduces the recognition sitefor T7 RNA Polymerase (5′ TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA 3′)(SEQ ID NO: 175) and 3′primer (5′ GCCTGTTGTGAGCCTCCTGTCGAA 3′) (SEQ IDNO: 176) results in the following template for transcription:

5′ TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA (26N)TTCGACAGGAGGCTCACAACAGGC 3′ (SEQ ID NO: 177)

The RNA transcript itself has the following sequence:

5′ GGGAGACAAGAAUAAACGCUCAA (26N) UUCGACAGGAGGCUCAC AACAGGC 3′ (SEQ IDNO: 178)Anti-E4 aptamers are selected using a conventional SELEX procedure asdescribed in U.S. Pat. No. 5,270,163. Each round consists of thefollowing steps:

-   1) Selection. The RNA and E4 protein are mixed, incubated at 37° C.,    washed through a nitrocellulose filter, and RNA is eluted from the    filters.-   2) Amplification. The RNA eluted from filters is extended with AMV    reverse transcriptase in the presence of 50 picomoles of 3′ primer    in a 50 μl reaction under conditions described in Gauss et al.    (1987). To the resulting cDNA synthesis 50 picomoles of 5′ primer is    added and in a reaction volume of 100 μl and amplified with Taq DNA    polymerase as described in Innis (1988) for 30 cycles.-   3) Transcription. In vitro transcription is performed on the    selected amplified templates as described in Milligan et al. (1987),    after which DNaseI is added to remove the DNA template. The    resultant selected RNA transcripts are then used in step 1 of the    next round. Only one-twentieth of the products created at each step    of the cycle are used in the subsequent cycles so that the history    of the selection can be traced. The progress of the selection method    is monitored by filter binding assays of labeled transcripts from    each PCR reaction. After the fourth round of selection and    amplification, the labeled selected RNA products produce binding to    E4. The binders are used in the detection of HPV in cells derived    from cervical smears.

EXAMPLE 5 Detection of HPV in Cutaneous and Mucosal Lesions

All the synthetic Fabs detect the HPV16 E1^E4 protein in formalin fixedparaffin-embedded tissue, although TVG405 consistently show the highestlevel of staining (FIG. 3).

FIG. 3 illustrates the use of synthetic Fabs to localise HPV16 E4protein in vivo by immunostaining of a low grade HPV16 CIN I with FabNIP-C11(Griffiths et al, 1994), which has no reactivity towards HPV16 E4(FIG. 3A), and the E4-specific Fab TVG405 which is described here (FIGS.3B, C, D). Fabs are detected using 9E10 as secondary antibody followedbv anti-mouse FITC conjugate. E4 is detectable in the upper layers ofthe epidermis but at greatly varying levels between different lesionswith often only a few positive cells being apparent (C, D). The positionof the basal layer is arrowed in C and D. Magnification is 200×.

Epitope exposure by microwave treatment enhances the sensitivity of E4detection, and even allows staining using TVG402 (Doorbar et al, 1992).The extent of E4 expression varies greatly between different lesions (8HPV16-associated CIN1 biopsies are examined), ranging from expressiononly in rare cells scattered throughout the biopsy (FIG. 3), towidespread distribution throughout the most differentiated layers of theepidermis (FIG. 4), comparable to the distribution of E4 in cutaneouswarts caused by HPV1 and HPV63 where the production of infectionsvirions is also high (FIG. 4). In low grade cervical intraepithelialneoplasia (CIN 1) caused by HPV16, E4 and L1 levels are also found tocorrelate closely, although expression of the two proteins is notcoincident (as previously suggested (Brown et al, 1994). E4 expressionprecedes the synthesis of the major capsid protein by several celllayers (as revealed by double staining, see FIG. 4) and in high gradecervical lesions (CIN 2/CIN 3) E4 is often abundant, even though theexpression of L1 is no longer supported (FIG. 4). This time delaybetween the commencement of E4 synthesis and the assembly of infectiousvirions is most apparent in HPV63, where E4 expression coincided withmigration of an infected basal cell into the parabasal layers, whileexpression of L1 is restricted to a narrow strip of cells in the uppergranular layer.

FIG. 4 demonstrates that synthesis of E4 is not directly linked to theexpression of capsid proteins in high and low grade HPV16 lesions, andbenign warts. FIG. 4 shows the results of triple staining using anti L1antisera (FIGS. 4A, D, G), HPV16 E4 Fab TVG405 (FIGS. 4B and 4E),polyclonal antisera to HPV63 E4 (FIG. 4H), and with DAPI (FIGS. 4C, F,I). A, B and C represent a low grade HPV16-induced lesion (CIN I). D, Eand F represent a high grade HPV16-induced lesion (CIN II/III). G, H andI represent a section through a verruca caused by HPV63. In all cases E4expression precedes L1 expression although by only a few cell layers inCIN I (A, B). In the CIN II/III we assume that terminal differentiationis insufficient to support synthesis of virion structural proteins (D)although E4 expression is abundant (E). The contrast between the onsetof E4 expression and the detection of virus structural proteins is mostapparent in cutaneous verrucas caused by HPV63 (G, H). The basal layeris indicated by an arrow on the DAPI-stained images. Magnification is100×.

Onset of Vegetative Viral DNA Replication and Expression of E4 Coincide

Vegetative viral DNA replication is found to begin in cells of the midspinous layer and to correlate exactly with the onset of E4 expression(FIG. 5).

FIG. 5 demonstrates that onset of vegetative viral DNA replicationcoincides with E4 expression in low grade HPV16 lesions and in benigncutaneous warts. The figure shows triple staining using the HPV16 E4antibodies TVG402, 405 and 406 (FIG. 5A) and HPV1 E4 antibodies 4.37 and9.95 (FIG. 5D), biotinylated DNA probe (FIG. 5B—HPV16. FIG. 5E—HPV1), orDAPI (FIGS. 5C and F). A, B and C represent a section through anHPV1-induced CIN I, and D, E and F represent a section through anHPV1-induced verruca. In the HPV16 CIN I, vegetative viral DNAreplication and E4 synthesis correlate in the mid to upper layers of theepidermis (A, B). In cutaneous lesions the two events are initiated assoon as the infected cell leaves the basal layer (D, E). Basal cells areillustrated in the DAPI counterstained image (F). Magnification is 200×.

In HPV 1-induced warts vegetative viral DNA replication and E4 synthesiscommence much earlier, and are evident immediately after the infectedbasal cell migrates into the superficial layers (FIG. 5). Only aproportion of the differentiating cells are permissive for vegetativeviral DNA replication, and only in these cells is E4 detectable.Neighbouring cells showed neither late gene expression nor vegetativeviral DNA replication, suggesting that onset of the two events isclosely linked. Although the sensitivity of DNA and E4 detection is notestablished, these ‘normal’ cells are likely to be either non-permissivefor viral replication or be uninfected. This precise correlation betweenE4 expression and the onset of vegetative viral DNA replication is alsoseen in cutaneous warts caused by HPV63 and 65, and in common wartscaused by HPV2.

Cells Undergoing Late Gene Expression Show an Abnormal Pattern ofTerminal Differentiation when Compared to Non-permissive or UninfectedCells

Cells supporting the late stages of HPV infection can thus be identifiedby immunostaining with Fab TVG405 (for HPV16) Mab 4.37 (for HPV1) orpolyclonal antisera to E4 (HPV63). In warts caused bv HPV1, E4-positivecells lack detectable levels of filaggrin or involucrin (FIG. 6(i)).Non-permissive (or uninfected) cells in the same lesion which showneither E4 expression nor vegetative viral DNA replication. expressfilaggrin and loricrin at levels indistinguishable from those in thesurrounding epidermis. Correlation of E4 synthesis with thedifferentiation-specific keratins K4 and K13 reveals an identicalpattern of inhibition. The intensity of K4 and K13 staining is alwayslower in E4-positive cells than in neighbouring cells that are notexpressing E4 (FIG. 6(ii)). K5 and 14, which are present in the basaland lower parabasal cells, are unaffected. This interference with thedetection of expression differentiation-specific keratins (K1 and K10 incutaneous skin) is also apparent in cutaneous warts caused by HPVI (FIG.6(ii)) but is not evident in warts caused by HPV63 (FIG. 6(ii)). The E4protein of HPV63 is most closely related to that of HPV1.

FIG. 6 illustrates that productive infection interferes with normalepithelial terminal differentiation. in low grade HPV16 lesions and inbenign cutaneous warts. FIG. 6(i) (keratin expression) shows triplestaining using the HPV16 E4 Fabs TVG405/TVG406 (FIG. 6(i)A), HPV 1 E4monoclonals 4.37/9.95 (D), and HPV63 E4 polyclonal antibodies (G), inconjunction with antibodies to the differentiation-specific mucosalkeratins 4 and 13 (B) or cutaneous keratins 1 and 10 (E, H). FIGS. 6(i)C, F and I show the DAPI counter stain. A, B and C represent a sectionthrough a HPV16-induced CIN I. D, E and F show a section through theedge of an HPV1-induced verruca, while FIGS. 6(i) G, H and I show asection through an HPV63-induced wart. In HPV16 and HPV1-inducedlesions, differentiation-specific keratins are less apparent inE4-positive cells than in neighbouring cells (A, B, D, E) although thisis not the case with HPV63 (G, H). Nuclear degeneration (visualised byDAPI counter staining) is retarded in E4-expressing cells (A, C, D, F).Magnification is 200×.

FIG. 6(ii) relates to filaggrin expression. The figure shows triplestaining, as described above, except that FIGS. 6(ii) B and E showfilaggrin staining. E4 staining is shown in FIGS. 6(ii) A and D, andDAPI counter staining is shown in FIGS. 6(ii) C and F. A, B and Crepresent the edge of an HPV63-induced wart where normal skin (left handside of figure) meets the benign tumour (right hand side of figure). D,E and F show the granular layer of an HPV1-induced wart. E4-positivecells do not express detectable levels of the differentiation-specificmarker filaggrin, and show marked nuclear preservation when compared toneighbouring uninfected or non-permissive cells. Magnification is 200×.

The Intracellular Distribution of the HPV16 E4 Proteins is Distinct fromthe Distribution of E4 in Cutaneous Lesions Caused by HPV1 and HPV63.

The E1^E4 protein of HPV1 is predominantly cytoplasmic and assemblesinto inclusions that coalesce and increase in size as the cell migratestowards the surface of the skin. The E1^E4 protein of HPV63 is found tohave a fibrous and granular distribution. By contrast, HPV16 E4 had afilamentous and perinuclear distribution in cells of the lower epidermallayers (FIG. 7), and assembled into prominent structures only in themore differentiated cell layers. These ‘inclusions’ are always foundsingly per cell (c.f. multiple inclusions found in most cutaneouslesions), are located adjacent to the nucleus, and are nearly alwaysdetected on the side of the nucleus closest to the surface of theepidermis. Although similar in appearance to the E4/intermediatefilament bundles which form after expression of the HPV16 E1^E4 proteinin epithelial cells in vitro, we have not detected the presence ofkeratins in these structures in vivo. Antibodies to the very N-terminusof HPV16 E1^E4 stained the structures much less readily than antibodiesto C-terminal epitopes (TVG 404, TVG405, TVG406) suggesting that theN-terminal region maybe either hidden or lost.

FIG. 7 shows the association of the HPV16 E4 proteins with perinuclearbundles and filamentous structure in vivo, in particular the detectionof HPV16 E4 proteins in the upper layers (FIGS. 7A, B) and lower layers(FIGS. 7C, D) of a HPV16 CIN I using a mixture of Fabs TVG405 andTVG406. In the upper layers E4 is diffuse throughout the cytoplasm butwith a prominent perinuclear pattern. Concentration of E4 intoperinuclear bundles (arrowed in FIG. 7B) is apparent in these cells. Inthe lower layers, E4 has a predominantly perinuclear and filamentousappearance (FIGS. 7C, D), but is not concentrated into perinuclearbundles. Magnification for FIGS. 7A and C is 200×; that for B and D is400×.

Confocal imaging revealed the N-terminal antibodies to localiseprimarily to the edge of the E4 structures while anti C-terminalstaining is strongest in the centre (data not shown). When compared tothe distribution seen with TVG405 and TVG406, the anti N-terminalreagent revealed HPV16 E1^E4 to have a more diffuse distribution in thecell (FIG. 8). No significant difference is apparent between thestaining pattern of TVG405, 406, 407 and the C-terminal antibody.

FIG. 8 provides evidence for processing of the HPV16 E4 proteins in vivoand shows triple staining in the upper layers of a HPV16 CIN using HPV16E4 Fab TVG406 which recognises an epitope in the C-terminal half of theE4 protein (FIG. 8A), an antibody to the N-terminal 12 amino acids ofthe HPV16 E1^E4 protein (FIG. 8B) and DAPI (FIG. 8C). TVG402, 403, 404,405 and 407 produced staining patterns that are not significantlydifferent from that of TVG 406. Anti N-terminal antibodies did noteffectively stain the perinuclear bundles (8B) which are evident withTVG406 (arrowed in 8A) suggesting that as in HPV1, different forms ofthe protein have different intracellular locations. Magnification is400×.

EXAMPLE 6 Detection of HSV in Cells Isolated from Cervical Lesions

Slides suitable for imaging of cells derived from cervical smearsstained using anti-E4 antibodies are prepared by the method set forth inU.S. Pat. No. 5,346,831. Cells are isolated from a patient according toconventional procedures and dissolved in 10 ml alcohol/saline buffer.The sample is prepared for centrifugation by disaggregating the clumpsor clusters of cells in the sample vial by vortexing. Afterdisaggregation, the sample is drained completely and layered over adensity gradient in a 12 ml conical cube, wherein the density gradientis formed with a plasma expander material comprising 6% betastarchsolution, and 0.9% physiological saline, also known by the tradename“Hespan” made by NPBI, Emmer-Compascuum, the Netherlands.

12 ml conical tubes containing density gradient and sample cells areplaced into centrifuge buckets, balance and centrifuged for 5 minutes,at a force of about 600 G. The liquid is then aspirated down to the 5 mlmark on the conical tube. The centrifuge buckets are removed and the 12ml conical tube centrifuged with remaining liquid for 10 minutes, at 800G. The tubes are emptied of supernatant, tapping lightly 2 or 3 times ata 45 degree angle. The tubes now contain packed cells of varyingvolumes. Upon mixing to homogeneity, the pellets generally contain thesame concentration of cells per unit volume of liquid.

50 μl of deionized H₂O is added, and the sample mixed by syringing 5times through a 0.042 inch tip. Upon completion of mixing, 150 μl ofsample followed by 500 μl of deionized H₂O is dispensed into asedimentation vessel attached to a slide which has been conventionallycoated with Poly-L lysine (1% Sigma). The transferred sample is allowedto settle within the vessel for approximately 10 minutes. The excesssample is aspirated off and the chamber rinsed with 2 ml deionized H₂Otwo times (aspirating between each addition).

FITC-labelled Fabs are then applied to the cells according to knownprocedures and the binding visualised by fluorescence microscopy.

References

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1. A method of diagnosing a precancerous lesion resulting from a mucosalpapilloma virus infection in an organism, the method comprisingdetecting vegetative mucosal papilloma viral DNA replication in a sampleof cells from said organism as indicated by mucosal papilloma virus-E4protein expression, wherein the detection of said virus-E4 proteincomprises the steps of: contacting in vitro a sample of the organism'scells from the site of potential infection with a molecule that bindsspecifically to mucosal papilloma virus-E4 protein; and monitoring saidbinding, wherein specific binding by said molecule to mucosal papillomavirus-E4 protein indicates the presence of mucosal papilloma virus-E4protein expression and the detection of vegetative mucosal papillomaviral DNA replication, thereby indicating a precancerous lesionresulting from a mucosal papilloma virus infection in an organism. 2.The method according to claim 1, wherein the organism is a mammal. 3.The method according to claim 2, wherein the organism is a human and thepapilloma virus is human papilloma virus (HPV).
 4. The method accordingto claim 2, wherein the site of potential infection is the cervix. 5.The method according to claim 3, wherein the human papilloma virus isselected from the group consisting of HPV types 16, 18, 33, 35, 45, 51,52, 56, 58 and
 61. 6. The method according to claim 1, furthercomprising determining the type(s) of HPV infection in the organism,said determination comprising the step of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 7. The method according to claim 1, wherein themolecule capable of binding to the papilloma virus E4 protein is capableof binding within a hydrophilic region of the E4 sequence.
 8. The methodaccording to claim 7, wherein the hydrophilic region is the region whichpossesses the sequence RPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) inHPV16. or its homologue in other papilloma viruses.
 9. The methodaccording to claim 8, wherein the hydrophilic region is the region whichpossesses the sequence RPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or itshomologue in other papilloma viruses.
 10. The method according to claim9, wherein the hydrophilic region is the region which possesses thesequence PKPSPWAPKKLH(R) (SEQ NO: 168) in HPV16, or its homologue inother papilloma viruses.
 11. The method according to claim 1, whereinthe molecule capable of binding to a papilloma virus E4 protein is anantibody or an antigen binding fragment thereof.
 12. The methodaccording to claim 3, wherein the site of potential infection is thecervix.
 13. The method according to claim 2, further comprisingdetermining the type(s) of HPV infection in the organism, saiddetermination comprising the steps of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 14. The method according to claim 3, furthercomprising determining the type(s) of HPV infection in the organism,said determination comprising the steps of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 15. The method according to claim 4, furthercomprising determining the type(s) of HPV infection in the organism,said determination comprising the steps of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 16. The method according to claim 5, furthercomprising determining the type(s) of HPV infection in the organism,said determination comprising the steps of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 17. The method according to claim 12, furthercomprising determining the type(s) of HPV infection in the organism,said determination comprising the steps of: contacting the cells with amolecule that binds specifically to a subset of HPV E4 proteins; andmonitoring said binding.
 18. The method according to claim 2, whereinthe molecule capable of binding to the papilloma virus E4 protein iscapable of binding within a hydrophilic region of the E4 sequence. 19.The method according to claim 3, wherein the molecule capable of bindingto the papilloma virus E4 protein is capable of binding within ahydrophilic region of the E4 sequence.
 20. The method according to claim4, wherein the molecule capable of binding to the papilloma virus E4protein is capable of binding within a hydrophilic region of the E4sequence.
 21. The method according to claim 5, wherein the moleculecapable of binding to the papilloma virus E4 protein is capable ofbinding within a hydrophilic region of the E4 sequence.
 22. The methodaccording to claim 6, wherein the molecule capable of binding to thepapilloma virus E4 protein is capable of binding within a hydrophilicregion of the E4 sequence.
 23. The method according to claim 12, whereinthe molecule capable of binding to the papilloma virus E4 protein iscapable of binding within a hydrophilic region of the E4 sequence. 24.The method according to claim 13, wherein the molecule capable ofbinding to the papilloma virus E4 protein is capable of binding within ahydrophilic region of the E4 sequence.
 25. The method according to claim14, wherein the molecule capable of binding to the papilloma virus E4protein is capable of binding within a hydrophilic region of the E4sequence.
 26. The method according to claim 15, wherein the moleculecapable of binding to the papilloma virus E4 protein is capable ofbinding within a hydrophilic region of the E4 sequence.
 27. The methodaccording to claim 16, wherein the molecule capable of binding to thepapilloma virus E4 protein is capable of binding within a hydrophilicregion of the E4 sequence.
 28. The method according to claim 17, whereinthe molecule capable of binding to the papilloma virus E4 protein iscapable of binding within a hydrophilic region of the E4 sequence. 29.The method according to claim 18, wherein the hydrophilic region is theregion which possesses the sequence RPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ IDNO:4) in HPV16. or its homologue in other papilloma viruses.
 30. Themethod according to claim 29, wherein the hydrophilic region is theregion which possesses the sequence RPIPKPSPWAPKKHR in HPV16 (SEQ IDNO:167), or its homologue in other papilloma viruses.
 31. The methodaccording to claim 30, wherein the hydrophilic region is the regionwhich possesses the sequence PKPSPWAPKKH(R) (SEQ NO:168) in HPV16, orits homologue in other papilloma viruses.
 32. The method according toclaim 19, wherein the hydrophilic region is the region which possessesthe sequence RPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or itshomologue in other papilloma viruses.
 33. The method according to claim32, wherein the hydrophilic region is the region which possesses thesequence RPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue inother papilloma viruses.
 34. The method according to claim 33, whereinthe hydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 35. The method according to claim 20, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 36. The method according to claim 35, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 37. The method according to claim 36, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 38. The method according to claim 21, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 39. The method according to claim 38, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 40. The method according to claim 39, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 41. The method according to claim 22, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 42. The method according to claim 41, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 43. The method according to claim 42, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 44. The method according to claim 23, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 45. The method according to claim 44, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 46. The method according to claim 45, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 47. The method according to claim 24, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 48. The method according to claim 47, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 49. The method according to claim 48, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 50. The method according to claim 25, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 51. The method according to claim 50, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 52. The method according to claim 51, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV 16, or its homologue in otherpapilloma viruses.
 53. The method according to claim 26, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 54. The method according to claim 53, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 55. The method according to claim 54, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 56. The method according to claim 27, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 57. The method according to claim 56, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 58. The method according to claim 57, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 59. The method according to claim 28, wherein thehydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) in HPV16. or its homologue inother papilloma viruses.
 60. The method according to claim 59, whereinthe hydrophilic region is the region which possesses the sequenceRPIPKPSPWAPKKHR in HPV16 (SEQ ID NO:167), or its homologue in otherpapilloma viruses.
 61. The method according to claim 60, wherein thehydrophilic region is the region which possesses the sequencePKPSPWAPKKH(R) (SEQ NO:168) in HPV16, or its homologue in otherpapilloma viruses.
 62. The method according to any one of claims 2–5,8–11, 14, 20–23, 25, 31–42, and 46–48, wherein the molecule capable ofbinding to a papilloma virus E4 protein is an antibody or an antigenbinding fragment thereof.